Dual specificity phosphatase 6 (DUSP6, also known as MKP3) and DUSP1 (MKP1) via allosteric inhibition. Inhibits ERK-stimulated DUSP6 activation. The compounds (including BCI and its analogs) inhibit DUSP6 and DUSP1 in the low micromolar range (IC₅₀ values in the micromolar range as shown in cellular complementation assays). Compound 7 (BCI-9) showed an EC₅₀ of 4.5 µM for FGF hyperactivation in zebrafish. Compound 19 (BCI-215) had an EC₅₀ of ~10 µM for FGF activation in zebrafish and inhibited DUSPs with IC₅₀ in the micromolar range. [1]

In RAW264.7 macrophages, BCI hydrochloride (100 ng/mL; 24 h) suppresses DUSP6 expression [2]. Lipopolysaccharide (LPS)-activated macrophages are inhibited in their expression by BCI hydrochloride (0-1 nM; 24 h). On the other hand, BCI hydrochloride (0-4 nM; 24 h) decreases ROS production and stimulates Nrf2 staining, which activates phagocytosis in LPS-activated macrophages[2].

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BCI and its analogs (e.g., 7 (BCI-9), 19 (BCI-215)) concentration-dependently increased pERK levels in DUSP1- or DUSP6-overexpressing HeLa cells in a chemical complementation assay, with IC₅₀ values in the micromolar range. [1]
In an in vitro phosphatase assay using recombinant DUSP6 and ERK2, BCI, 7, and 19 significantly suppressed ERK-stimulated activation of DUSP6, but did not inhibit basal phosphatase activity. The alcohol analog 31 (BCI-10), which lacked in vivo activity, showed insignificant suppression. [1]
In a cytotoxicity assay using EA.hy926 cells, BCI showed signs of cell loss, nuclear condensation, and necrosis (propidium iodide staining) at concentrations above 25 µM. In contrast, BCI-215 was devoid of cellular toxicity at concentrations up to 50 µM. [1]

In transgenic zebrafish embryos expressing GFP under the control of the dusp6 promoter (Tg(dusp6:EGFP)), BCI (20 µM) hyperactivated FGF signaling, quantified by increased GFP fluorescence in the head region using automated image analysis (Cognition Network Technology). Maximum GFP expression was observed after 5 hours of treatment. [1]
A series of 29 analogs were tested in this zebrafish model. Eleven compounds, including BCI, showed concentration-dependent hyperactivation of FGF signaling. Compound 7 (BCI-9) had the highest activity (EC₅₀ 4.5 µM). Structural features essential for in vivo activity included an aliphatic amino-alkyl side chain at C-3 and the α,β-unsaturated ketone moiety. [1]
Compound 19 (BCI-215) activated FGF signaling in vivo (EC₅₀ ~10 µM) but showed minimal whole-organism toxicity in zebrafish embryos after 24-hour exposure, even at twice its EC₅₀ (20 µM). Larvae treated with 19 hatched normally, whereas embryos treated with BCI or 7 at their EC₅₀ concentrations did not hatch. [1]
The in vivo FGF hyperactivating activity of active analogs correlated with their ability to inhibit DUSP6 and DUSP1 in mammalian cell-based assays. [1]

In vitro phosphatase activity was assessed using a 3-O-methylfluorescein phosphate (OMFP)-based assay. Recombinant His-tagged DUSP6 (250 ng) was pre-incubated with compounds (100 µM). To measure ERK2-stimulated DUSP6 activity, recombinant ERK2 (210 ng) was added to the DUSP6/compound mixture before initiating the reaction with OMFP (100 µM) in a final volume of 15 µL. Fluorescence (excitation/emission: 485/525 nm) was measured at 10-minute intervals for 1 hour at room temperature. This assay assessed the compounds' ability to inhibit the substrate (ERK2)-induced activation of DUSP6. [1]

Western Blot Analysis[2]
Cell Types: RAW264.7 Macrophage
Tested Concentrations: 100 ng/mL
Incubation Duration: 24 hrs (hours)
Experimental Results: demonstrated downregulation of DUSP6 protein.

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RT-PCR[2]
Cell Types: RAW264.7 Macrophage
Tested Concentrations: 0-1 nM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibits the expression of IL-1β and IL-6 mRNA in LPS-activated macrophages.

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DUSP inhibition was evaluated using a mammalian cell-based chemical complementation assay. HeLa cells were transfected with Myc-tagged DUSP1 or DUSP6 in 384-well plates. After 48 hours, cells were treated with compounds in quadruplicate wells for 15 minutes, followed by stimulation with phorbol ester (TPA, 500 ng/ml) for 15 minutes to activate the ERK pathway. Cells were immunostained with antibodies against phospho-ERK (pERK) and c-Myc. pERK and c-Myc-DUSP signals were visualized using fluorescent secondary antibodies. Plates were analyzed via multiparametric analysis. DUSP-expressing cells were identified based on c-Myc intensity. pERK levels in this subpopulation were quantified by comparing the cumulative pERK distribution of treated wells to vehicle-treated controls using Kolmogorov-Smirnov (KS) statistics. Higher KS values indicate greater restoration of pERK (i.e., DUSP inhibition). Dose-response curves were generated, and IC₅₀ values were calculated. [1]
Cytotoxicity was assessed in EA.hy926 cells. Cells were plated in 384-well plates, allowed to attach overnight, and treated with compound gradients for 6 hours. Cells were then stained with propidium iodide (PI, 1 µg/ml) and Hoechst 33342 (10 µg/ml) to label necrotic cells and nuclei, respectively. Live cell imaging was performed, and parameters like nuclei count, nuclear condensation, and percentage of PI-positive cells were determined. [1]

Zebrafish (*Tg(dusp6:EGFP)* embryos) were used for *in vivo* SAR and toxicity studies. Embryos were obtained by natural mating and incubated at 28.5°C. At 24 hours post-fertilization (hpf), individual embryos were placed into wells of a 96-well plate containing 200 µL of E3 embryo medium. Compounds were dissolved in DMSO as 100X stock solutions, and 2 µL was added directly to wells (final DMSO concentration 1%). For SAR studies, embryos were treated with compounds, and a negative control (DMSO) was included on each plate. After compound treatment (typically 5 hours for FGF activation assessment), embryos were anesthetized and imaged using a high-content reader with a 4X objective (excitation/emission: 488/525 nm). GFP expression in the head region was quantified using automated image analysis software. For toxicity assessment, after initial imaging, embryos were returned to the incubator and exposed to compounds for a total of 24 hours. Wells were then visually inspected for signs of toxicity such as gross morphological changes, necrosis, and impaired heartbeat or circulation. [1]

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Zebrafish (Tg(dusp6:EGFP) embryos) were used for in vivo SAR and toxicity studies. Embryos were obtained by natural mating and incubated at 28.5°C. At 24 hours post-fertilization (hpf), individual embryos were placed into wells of a 96-well plate containing 200 µL of E3 embryo medium. Compounds were dissolved in DMSO as 100X stock solutions, and 2 µL was added directly to wells (final DMSO concentration 1%). For SAR studies, embryos were treated with compounds, and a negative control (DMSO) was included on each plate. After compound treatment (typically 5 hours for FGF activation assessment), embryos were anesthetized and imaged using a high-content reader with a 4X objective (excitation/emission: 488/525 nm). GFP expression in the head region was quantified using automated image analysis software. For toxicity assessment, after initial imaging, embryos were returned to the incubator and exposed to compounds for a total of 24 hours. Wells were then visually inspected for signs of toxicity such as gross morphological changes, necrosis, and impaired heartbeat or circulation. [1]

In zebrafish embryos, many active analogs, including BCI, showed whole-organism toxicity at higher doses, manifested as gross morphological changes, bent tail phenotype, and the appearance of opaque, necrotic cells after 24-hour exposure. Toxicity was confirmed by acridine orange staining revealing dead cells in the tail. [1]
However, toxicity did not correlate directly with the electrophilicity (measured by Hammett σ constants) of the α,β-unsaturated ketone moiety or with in vivo target activity. Some inactive analogs were also toxic. [1]
Compound 19 (BCI-215) was identified as a non-toxic analog. It showed no toxicity at concentrations twice its EC₅₀ for FGF activation (20 µM). Larvae treated with 19 hatched normally by 56 hpf, unlike those treated with BCI or 7. [1]
In vitro cytotoxicity in EA.hy926 cells recapitulated the differential toxicity: BCI showed toxicity above 25 µM, while BCI-215 was non-toxic up to 50 µM. [1]

[1]. In vivo structure-activity relationship studies support allosteric targeting of a dual specificity phosphatase. Chembiochem. 2014 Jul 7;15(10):1436-45.

[2]. DUSP6 Inhibitor (E/Z)-BCI Hydrochloride Attenuates Lipopolysaccharide-Induced Inflammatory Responses in Murine Macrophage Cells via Activating the Nrf2 Signaling Axis and Inhibiting the NF-κB Pathway. Inflammation. 2019 Apr;42(2):672-681.

BCI was discovered as an allosteric inhibitor of DUSP6 via a phenotypic screen in zebrafish, highlighting the utility of zebrafish for in vivo SAR studies. It inhibits DUSP6 by binding to a novel allosteric site adjacent to the phosphatase active site, preventing the conformational change required for ERK-stimulated activation. Molecular modeling suggests the cyclohexylamino side chain and the α,β-unsaturated ketone of BCI form hydrogen bonds with Arg299 and Trp264 of DUSP6, respectively. The alcohol analog 31, lacking the ketone, cannot form this key hydrogen bond and is inactive. [1]
The study demonstrates that DUSPs can be targeted via allosteric mechanisms, circumventing challenges posed by their shallow, conserved active sites and redox-sensitive catalytic cysteine. BCI and its analogs inhibit both DUSP6 and DUSP1, suggesting a lack of selectivity between these two DUSPs. Compound 19 (BCI-215), with potent DUSP inhibitory activity and minimal toxicity, is proposed as an improved chemical probe for studying DUSP1/DUSP6 biology. [1]

352.147

95130-23-7

BCI;1245792-51-1;(E/Z)-BCI;15982-84-0

20831631

Light yellow to yellow solid powder

484.6ºC at 760mmHg

161.3ºC

2.324

2

2

3

25

470

0

Cl.O=C1C(=CC2C=CC=CC=2)C(NC2CCCCC2)C2C1=CC=CC=2

JPATUDRDKCLPTI-CRDKNBMZSA-N

InChI=1S/C22H23NO.ClH/c24-22-19-14-8-7-13-18(19)21(23-17-11-5-2-6-12-17)20(22)15-16-9-3-1-4-10-16/h1,3-4,7-10,13-15,17,21,23H,2,5-6,11-12H21H/b20-15-

2-Benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one hydrochloride

BCI hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~15.62 mg/mL (~44.14 mM)

Solubility in Formulation 1: ≥ 1.56 mg/mL (4.41 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 15.6 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.56 mg/mL (4.41 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 15.6 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1.56 mg/mL (4.41 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 15.6 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)